A phenotypic screening platform for identifying chemical modulators of astrocyte reactivity.

Disease, injury and aging induce pathological reactive astrocyte states that contribute to neurodegeneration. Modulating reactive astrocytes therefore represent an attractive therapeutic strategy. Here we describe the development of an astrocyte phenotypic screening platform for identifying chemical modulators of astrocyte reactivity. Leveraging this platform for chemical screening, we identify histone deacetylase 3 (HDAC3) inhibitors as effective suppressors of pathological astrocyte reactivity. We demonstrate that HDAC3 inhibition reduces molecular and functional characteristics of reactive astrocytes in vitro. Transcriptional and chromatin mapping studies show that HDAC3 inhibition disarms pathological astrocyte gene expression and function while promoting the expression of genes associated with beneficial astrocytes. Administration of RGFP966, a small molecule HDAC3 inhibitor, blocks reactive astrocyte formation and promotes neuroprotection in vivo in mice. Collectively, these results establish a platform for discovering modulators of reactive astrocyte states, inform the mechanisms that control astrocyte reactivity and demonstrate the therapeutic benefits of modulating astrocyte reactivity for neurodegenerative diseases.

Benefit of clinical pharmacists in neurology clinics at an academic medical center.

BACKGROUND: Patients with neurologic diseases have complex medical needs and may benefit from the addition of clinical pharmacists in their care. OBJECTIVES: This study aimed to describe integration and benefit of clinical pharmacists in neuroimmunology and neuromuscular clinics at an academic medical center. METHODS: This retrospective chart review evaluated patients initiated on a neurology medication for a neuroimmunology or neuromuscular disease state before and after pharmacist integration in neurology clinics. The primary outcome measured access to an initially prescribed neuroimmunology or neuromuscular medication within 90 days of prescription. Secondary outcomes included access to an initially prescribed or alternative neurology medication owing to insurance requirements within 90 days, time from initial prescription to start, and description of pharmacist involvement. RESULTS: There were 101 patients in the pregroup and 101 patients in the postgroup. The percentage of patients with confirmed initially prescribed medication access at 90 days increased in the postgroup compared with the pregroup (87.1% vs. 72.5%, respectively, P = 0.014). For secondary outcomes, the percentage of patients who started on an initially prescribed or alternative neuroimmunology or neuromuscular medication within 90 days also increased in the postgroup compared with the pregroup (90.0% vs. 73.3%, respectively, P = 0.004). Additional pharmacist involvement occurred in 64 patients (63.4%) in the postgroup and included prior authorization approval assistance, drug information support, and medication liaison interventions, with an average of 4.7 pharmacist interventions at each pharmacy-led encounter. CONCLUSION: The addition of pharmacists into neuroimmunology and neuromuscular clinics improved operational access to medications for neuroimmunology and neuromuscular conditions. In addition, pharmacists were able to assist with multiple areas of patient care including medication education, monitoring, and serving as a medication liaison. This study supports continuing to offer clinical pharmacy services in neuroimmunology and neuromuscular departments and may support the addition of clinical pharmacists into neurology services at other institutions.

Seven Strategies to Integrate Equity within Translational Research in Neurology.

The rapidly accelerating translation of biomedical advances is leading to revolutionary therapies that are often inaccessible to historically marginalized populations. We identified and synthesized recent guidelines and statements to propose 7 strategies to integrate equity within translational research in neurology: (1) learn history; (2) learn about upstream forces; (3) diversify and liberate; (4) change narratives and adopt best communication practices; (5) study social drivers of health and lived experiences; (6) leverage health technologies; and (7) build, sustain, and lead culturally humble teams. We propose that equity should be a major goal of translational research, equally important as safety and efficacy. ANN NEUROL 2024;95:432-441.

Cytokine polarized, alternatively activated bone marrow neutrophils drive axon regeneration.

The adult central nervous system (CNS) possesses a limited capacity for self-repair. Severed CNS axons typically fail to regrow. There is an unmet need for treatments designed to enhance neuronal viability, facilitate axon regeneration, and ultimately restore lost neurological functions to individuals affected by traumatic CNS injury, multiple sclerosis, stroke, and other neurological disorders. Here we demonstrate that both mouse and human bone marrow (BM) neutrophils, when polarized with a combination of recombinant interleukin (IL)-4 and granulocyte-colony stimulating factor (G-CSF), upregulate alternative activation markers and produce an array of growth factors, thereby gaining the capacity to promote neurite outgrowth. Moreover, adoptive transfer of IL-4/G-CSF polarized BM neutrophils into experimental models of CNS injury triggered substantial axon regeneration within the optic nerve and spinal cord. These findings have far-reaching implications for the future development of autologous myeloid cell-based therapies that may bring us closer to effective solutions for reversing CNS damage.

Biological aging in multiple sclerosis.

Multiple sclerosis (MS) is most likely to adopt a progressive clinical course during middle age or beyond, and the number of older adults with MS is steadily increasing. Developing new strategies to manage progressive forms of MS, which do not respond to currently available disease-modifying therapies (DMTs), will require a deeper understanding of the mechanisms by which biological aging interacts with pathogenic pathways to propel disability accumulation. In experimental autoimmune encephalomyelitis (EAE), a widely used preclinical mouse model of MS, middle-aged animals experience a more severe and protracted clinical course than their younger counterparts. This exacerbated disease course is accompanied by persistent neuroinflammation. Clinical studies of age-related biomarkers, such as telomere length, senescence markers, and DNA methylation, suggest that biological aging is accelerated in people with MS compared with age- and sex-matched healthy controls. Furthermore, distinguishing biological age from chronological may afford more precision in determining aging effects in MS. Here we review the current literature on aging biology and its impact on MS pathogenesis. Future research on this topic may lead to the development of novel biomarkers and senotherapy agents that slow neurological decline in people with progressive MS by targeting relevant aging-related pathways.

Transverse myelitis associated with Mpox infection.

Neurologic manifestations of mpox (monkeypox) infection are common. Rarely, transverse myelitis has been associated with mpox infection. We describe a case of longitudinally extensive transverse myelitis in a patient with recently diagnosed mpox, presenting as acute flaccid paraplegia. The patient underwent an extensive work-up that included serological and cerebrospinal fluid (CSF) testing and magnetic resonance imaging (MRI). They were treated with tecoviromat, high dose steroids, and intravenous immunoglobulin, followed by plasma exchange. Despite these interventions, there was minimal neurologic improvement. This case underscores the importance of instituting measures designed to prevent mpox infection, including public education initiatives.

Respiratory tract Moraxella catarrhalis and Klebsiella pneumoniae can promote pathogenicity of myelin-reactive Th17 cells.

The respiratory tract is home to a diverse microbial community whose influence on local and systemic immune responses is only beginning to be appreciated. The airways have been linked with the trafficking of myelin-specific T-cells in the preclinical stages of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Th17 cells are important pathogenic effectors in MS and EAE but are innocuous immediately following differentiation. Upregulation of the cytokine GM-CSF appears to be a critical step in their acquisition of pathogenic potential, but little is known about the mechanisms that mediate this process. Here, primed myelin-specific Th17 cells were transferred to congenic recipient mice prior to exposure to various human respiratory tract-associated bacteria and T-cell trafficking, phenotype and the severity of resulting EAE were monitored. Disease was exacerbated in mice exposed to the Proteobacteria Moraxella catarrhalis and Klebsiella pneumoniae, but not the Firmicute Veillonella parvula, and this was associated with significantly increased GM-CSF+ and GM-CSF+IFNγ+ ex-Th17-like donor CD4 T cells in the lungs and central nervous system (CNS) of these mice. These findings support the concept that respiratory bacteria may contribute to the pathophysiology of CNS autoimmunity by modulating pathogenicity in crucial T-cell subsets that orchestrate neuroinflammation.

TAM receptor signaling dictates lesion location and clinical phenotype during experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), induced by the adoptive transfer of Th17 cells, typically presents with ascending paralysis and inflammatory demyelination of the spinal cord. Brain white matter is relatively spared. Here we show that treatment of Th17 transfer recipients with a highly selective inhibitor to the TAM family of tyrosine kinase receptors results in ataxia associated with a shift of the inflammatory infiltrate to the hindbrain parenchyma. During homeostasis and preclinical EAE, hindbrain microglia express high levels of the TAM receptor Mer. Our data suggest that constitutive TAM receptor signaling in hindbrain microglia confers region-specific protection against Th17 mediated EAE.

Ageing and multiple sclerosis.

The factor that is most relevant and strongly associated with the clinical course of multiple sclerosis is chronological age. Very young patients exclusively have relapsing remitting disease, whereas those with later onset disease face a more rapid development of permanent disability. For people with progressive multiple sclerosis, the poor response to current disease modifying therapies might be related to ageing in the immune system and CNS. Ageing is also associated with increased risks of side-effects caused by some multiple sclerosis therapies. Both somatic and reproductive ageing processes might contribute to development of progressive multiple sclerosis. Understanding the role of ageing in immune and neural cell function in patients with multiple sclerosis might be key to halting non-relapse-related progression. The growing literature on potential therapies that target senescent cells and ageing processes might provide effective strategies for remyelination and neuroprotection.

Characterization of Zymosan-Modulated Neutrophils With Neuroregenerative Properties.

Recent studies using advanced techniques such as single cell RNA sequencing (scRNAseq), high parameter flow cytometry, and proteomics reveal that neutrophils are more heterogeneous than previously appreciated. Unique subsets have been identified in the context of bacterial and parasitic infections, cancer, and tissue injury and repair. The characteristics of infiltrating neutrophils differ depending on the nature of the inflammation-inciting stimulus, the stage of the inflammatory response, as well as the tissue microenvironment in which they accumulate. We previously described a new subpopulation of immature Ly6Glow neutrophils that accumulate in the peritoneal cavity 3 days following intraperitoneal (i.p.) administration of the fungal cell wall extract, zymosan. These neutrophils express markers of alternative activation and possess neuroprotective/regenerative properties. In addition to inducing neurite outgrowth of explanted neurons, they enhance neuronal survival and axon regeneration in vivo following traumatic injury to the optic nerve or spinal cord. In contrast, the majority of neutrophils that accumulate in the peritoneal fluid 4 hours following i.p. zymosan injection (4h NΦ) have features of conventional, mature Ly6Ghi neutrophils and lack neuroprotective or neuroregenerative properties. In the current study, we expand upon on our previously published observations by performing a granular, in-depth analysis of these i.p. zymosan-modulated neutrophil populations using scRNAseq and high parameter flow cytometry. We also analyze cell lysates of each neutrophil population by liquid chromatography/mass spectrometry. Circulating blood neutrophils, harvested from naive mice, are analyzed in parallel as a control. When samples were pooled from all three groups, scRNAseq revealed 11 distinct neutrophil clusters. Pathway analyses demonstrated that 3d NΦ upregulate genes involved in tissue development and wound healing, while 4h NΦ upregulate genes involved in cytokine production and perpetuation of the immune response. Proteomics analysis revealed that 3d NΦ and 4h NΦ also express distinct protein signatures. Adding to our earlier findings, 3d NΦ expressed a number of neuroprotective/neuroregenerative candidate proteins that may contribute to their biological functions. Collectively, the data generated by the current study add to the growing literature on neutrophil heterogeneity and functional sub-specialization and might provide new insights in elucidating the mechanisms of action of pro-regenerative, neuroprotective neutrophil subsets.

Biological aging of CNS-resident cells alters the clinical course and immunopathology of autoimmune demyelinating disease.

Biological aging is the strongest factor associated with the clinical phenotype of multiple sclerosis (MS). Relapsing-remitting MS typically presents in the third or fourth decade, whereas the mean age of presentation of progressive MS (PMS) is 45 years old. Here, we show that experimental autoimmune encephalomyelitis (EAE), induced by the adoptive transfer of encephalitogenic CD4+ Th17 cells, was more severe, and less likely to remit, in middle-aged compared with young adult mice. Donor T cells and neutrophils were more abundant, while B cells were relatively sparse, in CNS infiltrates of the older mice. Experiments with reciprocal bone marrow chimeras demonstrated that radio-resistant, nonhematopoietic cells played a dominant role in shaping age-dependent features of the neuroinflammatory response, as well as the clinical course, during EAE. Reminiscent of PMS, EAE in middle-aged adoptive transfer recipients was characterized by widespread microglial activation. Microglia from older mice expressed a distinctive transcriptomic profile suggestive of enhanced chemokine synthesis and antigen presentation. Collectively, our findings suggest that drugs that suppress microglial activation, and acquisition or expression of aging-associated properties, may be beneficial in the treatment of progressive forms of inflammatory demyelinating disease.

Neutralizing antibody responses against SARS-CoV-2 in vaccinated people with multiple sclerosis.

Background: Patients with multiple sclerosis (pwMS) are often treated with disease modifying therapies (DMT) with immunomodulatory effects. This is of particular concern following the development of several vaccines to combat coronavirus disease 19 (COVD-19), a potentially fatal illness caused by SARS-CoV-2. Objectives: To determine the efficacy of SARS-CoV-2 vaccination in pwMS and the impact of disease modifying therapies (DMT) on vaccine response. Methods: This is a prospective longitudinal study in pwMS. Longitudinal serum samples were obtained prior to, and after SARS-CoV-2 mRNA vaccination. A novel neutralizing antibody (nAb) assay was used to determine nAbs titres against SARS-CoV-2 spike. Results: We observed that (1) pwMS on B-cell depleting therapies exhibited reduced response to vaccination compared to other pwMS, correlating with time from last anti-CD20 infusion, (2) prior COVID-19 illness, DMT category, and pyramidal function were significant predictors of vaccine responsiveness, and (3) circulating absolute lymphocyte count (ALC) and IgG levels correlated with nAb levels. Conclusions: We demonstrate that pwMS exhibit reduced nAb response to mRNA vaccination dependent on DMT status and identify predictive biomarkers for vaccine efficacy. We conclude that additional vaccination strategies may be necessary to achieve protective immunity in pwMS.

Encephalitogenic and Regulatory CD8 T Cells in Multiple Sclerosis and Its Animal Models.

Multiple sclerosis (MS), a neuroinflammatory disease that affects millions worldwide, is widely thought to be autoimmune in etiology. Historically, research into MS pathogenesis has focused on autoreactive CD4 T cells because of their critical role in the animal model, experimental autoimmune encephalomyelitis, and the association between MS susceptibility and single-nucleotide polymorphisms in the MHC class II region. However, recent studies have revealed prominent clonal expansions of CD8 T cells within the CNS during MS. In this paper, we review the literature on CD8 T cells in MS, with an emphasis on their potential effector and regulatory properties. We discuss the impact of disease modifying therapies, currently prescribed to reduce MS relapse rates, on CD8 T cell frequency and function. A deeper understanding of the role of CD8 T cells in MS may lead to the development of more effective and selective immunomodulatory drugs for particular subsets of patients.

Analysis of the immune response to sciatic nerve injury identifies efferocytosis as a key mechanism of nerve debridement.

Sciatic nerve crush injury triggers sterile inflammation within the distal nerve and axotomized dorsal root ganglia (DRGs). Granulocytes and pro-inflammatory Ly6Chigh monocytes infiltrate the nerve first and rapidly give way to Ly6Cnegative inflammation-resolving macrophages. In axotomized DRGs, few hematogenous leukocytes are detected and resident macrophages acquire a ramified morphology. Single-cell RNA-sequencing of injured sciatic nerve identifies five macrophage subpopulations, repair Schwann cells, and mesenchymal precursor cells. Macrophages at the nerve crush site are molecularly distinct from macrophages associated with Wallerian degeneration. In the injured nerve, macrophages 'eat' apoptotic leukocytes, a process called efferocytosis, and thereby promote an anti-inflammatory milieu. Myeloid cells in the injured nerve, but not axotomized DRGs, strongly express receptors for the cytokine GM-CSF. In GM-CSF-deficient (Csf2-/-) mice, inflammation resolution is delayed and conditioning-lesion-induced regeneration of DRG neuron central axons is abolished. Thus, carefully orchestrated inflammation resolution in the nerve is required for conditioning-lesion-induced neurorepair.

A new neutrophil subset promotes CNS neuron survival and axon regeneration.

Transected axons typically fail to regenerate in the central nervous system (CNS), resulting in chronic neurological disability in individuals with traumatic brain or spinal cord injury, glaucoma and ischemia-reperfusion injury of the eye. Although neuroinflammation is often depicted as detrimental, there is growing evidence that alternatively activated, reparative leukocyte subsets and their products can be deployed to improve neurological outcomes. In the current study, we identify a unique granulocyte subset, with characteristics of an immature neutrophil, that had neuroprotective properties and drove CNS axon regeneration in vivo, in part via secretion of a cocktail of growth factors. This pro-regenerative neutrophil promoted repair in the optic nerve and spinal cord, demonstrating its relevance across CNS compartments and neuronal populations. Our findings could ultimately lead to the development of new immunotherapies that reverse CNS damage and restore lost neurological function across a spectrum of diseases.

Chemokine receptor CXCR3 is required for lethal brain pathology but not pathogen clearance during cryptococcal meningoencephalitis.

Cryptococcal meningoencephalitis (CM) is the major cause of infection-related neurological death, typically seen in immunocompromised patients. However, T cell-driven inflammatory response has been increasingly implicated in lethal central nervous system (CNS) immunopathology in human patients and murine models. Here, we report marked up-regulation of the chemokine receptor CXCR3 axis in human patients and mice with CM. CXCR3 deletion in mice improves survival, diminishes neurological deficits, and limits neuronal damage without suppressing fungal clearance. CD4+ T cell accumulation and TH1 skewing are reduced in the CNS but not spleens of infected CXCR3-/- mice. Adoptive transfer of WT, but not CXCR3-/- CD4+ T cells, into CXCR3-/- mice phenocopies the pathology of infected WT mice. Collectively, we found that CXCR3+CD4+ T cells drive lethal CNS pathology but are not required for fungal clearance during CM. The CXCR3 pathway shows potential as a therapeutic target or for biomarker discovery to limit CNS inflammatory damages.

Mature myelin maintenance requires Qki to coactivate PPARß-RXRα-mediated lipid metabolism.

Lipid-rich myelin forms electrically insulating, axon-wrapping multilayers that are essential for neural function, and mature myelin is traditionally considered metabolically inert. Surprisingly, we discovered that mature myelin lipids undergo rapid turnover, and quaking (Qki) is a major regulator of myelin lipid homeostasis. Oligodendrocyte-specific Qki depletion, without affecting oligodendrocyte survival, resulted in rapid demyelination, within 1 week, and gradually neurological deficits in adult mice. Myelin lipids, especially the monounsaturated fatty acids and very-long-chain fatty acids, were dramatically reduced by Qki depletion, whereas the major myelin proteins remained intact, and the demyelinating phenotypes of Qki-depleted mice were alleviated by a high-fat diet. Mechanistically, Qki serves as a coactivator of the PPARß-RXRα complex, which controls the transcription of lipid-metabolism genes, particularly those involved in fatty acid desaturation and elongation. Treatment of Qki-depleted mice with PPARß/RXR agonists significantly alleviated neurological disability and extended survival durations. Furthermore, a subset of lesions from patients with primary progressive multiple sclerosis were characterized by preferential reductions in myelin lipid contents, activities of various lipid metabolism pathways, and expression level of QKI-5 in human oligodendrocytes. Together, our results demonstrate that continuous lipid synthesis is indispensable for mature myelin maintenance and highlight an underappreciated role of lipid metabolism in demyelinating diseases.

Multiple sclerosis relapse risk in the postoperative period: Effects of invasive surgery and anesthesia.

BACKGROUND: Postoperative multiple sclerosis (MS) relapses are a concern among patients and providers. OBJECTIVE: To determine whether MS relapse risk is higher postoperatively. METHODS: Data were extracted from medical records of MS patients undergoing surgery at a tertiary center (2000-2016). Conditional logistic regression estimated within-patient unadjusted and age-adjusted odds of postoperative versus preoperative relapse. RESULTS: Among 281 patients and 609 surgeries, 12 postoperative relapses were identified. The odds of postoperative versus preoperative relapse in unadjusted (odds ratio (OR) = 0.56, 95% confidence interval (CI) = 0.18-1.79; p = 0.33) or age-adjusted models (OR = 0.66, 95% CI = 0.20-2.16; p = 0.49) were not increased. CONCLUSIONS: Surgery/anesthesia exposure did not increase postoperative relapse risk. These findings require confirmation in larger studies.